This disclosure relates generally to the field of immobilized proteins and is specifically concerned with a novel method of attaching usefully active proteins such as enzymes to the surfaces of a variety of inorganic support materials.
The desirability of attaching useful proteins such as enzymes, antibodies, and the like onto insoluble support materials is well known. In general the immobilization of such biologically active materials results in insoluble composite materials which can be reused and/or easily separated from a reaction medium.
A wide variety of materials has been used successfully as support or carrier materials for proteins. The carriers may be organic (e.g. U.S. Pat. No. 3,645,852), inorganic (e.g. U.S. Pat. No. 3,556,945), or a combination of the two (e.g. 3,705,084). Various modes of attachment include simple adsorption, entrapment, and chemical coupling of the proteins to the carrier surfaces.
Recent studies have shown that for many applications, inorganic carriers are preferable to organic materials and the various advantages are described and/or demonstrated in several relatively recent patents (e.g. U.S. Pat. No. 3,556,945; describing the adsorption of enzymes to porous glass; U.S. Pat. No. 3,519,538, describing the covalent bonding of enzymes to silanized inorganics; U.S. Pat. No. 3,562,761, describing the covalent bonding of antibodies to silanized inorganics; and U.S. Pat. No. 3,850,751, describing the adsorption of enzymes to a variety of non-siliceous inorganics such as alumina, titania, and the like).
Much of the early work with inorganic carriers involved using those materials to simply adsorb proteins from a solution. Although that technique of bonding has many obvious advantages, it was apparent that the actual bonding forces were relatively weak and pH dependent, thus limiting the use of adsorbed protein systems. It should be noted, however, that some adsorbed enzyme systems have been shown to be highly useful. See, for example, U.S. Pat. No. 3,868,304, disclosing the use of glucose isomerase adsorbed to highly porous alumina particles to isomerize glucose to fructose.
In an effort to remedy some of the disadvantages of adsorbed enzymes, attempts were made to covalently bond non-essential portions of protein molecules to various carriers. In the case of inorganic supports, it was found that silane coupling agents could be used as an intermediate link between the inorganic surface and the protein (see U.S. Pat. No. 3,519,538, enzymes, and U.S. Pat. No. 3,652,761, antibodies). The resulting composites possessed the known advantages of inorganic carriers and demonstrated a relatively stronger bonding of the proteins. Although silane coupling agents can be used to bond a wide variety of materials to inorganics, they are relatively few in number and, in some cases, relatively expensive. Also, their use requires several processing steps. For example, after attachment to an inorganic surface (e.g., glass) it is often necessary, depending on the silane used, to further functionalize the silane to render it suitable for covalent bonding.
Surprisingly, I have found that proteins can be attached to inorganic carriers by means of an adsorbed surface derivative. To the adsorbed surface derivative, a variety of proteins can be covalently bonded without loss of biological activity. Thus, the relative ease of adsorbing the coupling agent to an inorganic to produce a relatively strongly bonded surface derivative is combined with being able to subsequently bond a protein via covalent bonds to the derivatized surface. Details of the method are described herein.